Assessment of Drug Proarrhythmic Potential in Electrically Paced Human Induced Pluripotent Stem Cell–Derived Ventricular Cardiomyocytes Using Multielectrode Array

Abstract

Human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) have been widely used for the assessment of drug proarrhythmic potential through multielectrode array (MEA). HiPSC-CM cultures beat spontaneously with a wide range of frequencies, however, which could affect drug-induced changes in repolarization. Pacing hiPSC-CMs at a physiological heart rate more closely resembles the state of in vivo ventricular myocytes and permits the standardization of test conditions to improve consistency. In this study, we systematically investigated the time window of stable ion currents in high-purity hiPSC-derived ventricular cardiomyocytes (hiPSC-vCMs) and confirmed that these cells could be used to correctly predict the proarrhythmic risk of Comprehensive In Vitro Proarrhythmia Assay (CiPA) reference compounds. To evaluate drug proarrhythmic potentials at a physiological beating rate, we used a MEA to electrically pace hiPSC-vCMs, and we recorded regular field potential waveforms in hiPSC-vCMs treated with DMSO and 10 CiPA reference drugs. Prolongation of field potential duration was detected in cells after exposure to high- and intermediate-risk drugs; in addition, drug-induced arrhythmia-like events were observed. The results of this study provide a simple and feasible method to investigate drug proarrhythmic potentials in hiPSC-CMs at a physiological beating rate.

Keywords

human induced pluripotent stem cell–derived ventricular cardiomyocytes (hiPSC-vCMs)cardiac safety assay electrical pacing microelectrode array (MEA)Comprehensive In Vitro Proarrhythmia Assay (CiPA)

Introduction

The Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative proposes use of human stem cell–derived cardiomyocytes to validate the proarrhythmic risks [e.g., torsades de pointes (TdP)] of new chemical entities.¹ Although CiPA validation results for reference compounds in spontaneously beating human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) are promising,² the purity and spontaneous beating of ventricular cardiomyocytes, two important aspects of hiPSC-CMs with regard to drug cardiac safety assessment, should be considered. Typically, differentiated hiPSC-CMs comprise mixtures of ventricular-like (approximately 60–70%), atrial-like, and nodal-like cardiomyocytes, which have different electrophysiological characteristics^3,4 that may cause variable results of drug effects on human pluripotent stem cell–derived cardiomyocyte (hPSC-CM) preparations. HiPSC-CM cultures spontaneously beat at various rates influenced by many factors, such as different differentiation protocols and cell sources,^3–7 whereas in vivo adult ventricular myocardium beats under the control of the sinoatrial node. Beating rate is an important determinant of the field potential duration (FPD) of hiPSC-CMs, and different beating rates can affect both FPD and changes in drug-induced repolarization duration, as measured with a multielectrode array (MEA) system.⁸ Examining the effects of drugs on hiPSC-CMs in conditions similar to the in vivo environment is expected to eliminate variations in testing caused by discrepancies of beating rates. Compared with optical stimulation,⁸ electrical pacing is simpler and does not require transfection of light-sensitive protein (e.g., channel rhodopsin). The “T” waves in field potential (FP) signals corresponding to the ventricular repolarization in electrocardiogram, however, are too tiny to be detected under electrical pacing, which makes its application in assessment of drug proarrhythmic potential difficult.⁹

Previously, we obtained highly homogeneous (>90%) hiPSC-derived ventricular cardiomyocytes (hiPSC-vCMs) using a direct ventricular differentiation protocol with chemically defined and albumin-free conditions.⁷ Without fetal bovine serum or large amounts of albumin in the media, the efficiency and yield of cardiac differentiation were more consistent between batches. Due to the high purity of ventricular cardiomyocytes, these cells could be a good tool for cardiac safety evaluation. In addition, without the impact of the unknown component in the serum or albumin, the differentiation process can be further improved to better accommodate cardiac safety evaluation needs.

In this study, we systematically investigated the time window of stable ion currents of differentiated hiPSC-vCMs, and demonstrated that hiPSC-vCMs correctly predicted the proarrhythmic risk of CiPA reference compounds. Although these cells exhibited spontaneous beating, the beating rate was so slow that electrical pacing with a physiological rate (1~1.2 Hz) was feasible. Thus, we established a simple electrical pacing method that can be used for the assessment of drug proarrhythmic risk.

Materials and Methods

Cell Source

HiPSC-vCMs were differentiated according to a previously reported method.⁷ The hiPSC-vCMs at day 30 after cardiac induction were cryopreserved as Cauliscell hiPSC-vCMs (cat. no. 300106; Cauliscell Company, Nanjing, China). The purity of cardiomyocytes was 97.3 ± 0.8% ( Suppl. Fig. S1A ).

Manual Patch Clamp Recording

At the designated time points, beating colonies were dissociated into single cells according to a previously reported protocol.¹⁰ Spontaneous action potentials (APs) and ionic currents were recorded at 35 ± 2 °C except for calcium current (I_Ca) measurements, which were performed at room temperature. Action potential, sodium current (I_Na), I_Ca, and E-4031-sensitive potassium current (I_Kr) recordings were carried out on single hiPSC-vCMs, as reported previously.⁷ Perforated patch recording was used for pharmacological examinations of stimulated APs.⁷

MEA Recordings

Cell culture

The cryopreserved hiPSC-vCMs were used in this study. After thawing, hiPSC-vCMs were plated into human recombinant vitronectin-coated six-well plates in 2 mL of cardiomyocyte plating medium at a density of 1 × 10⁶ viable cells/mL. Twenty-four hours post thaw, the medium was replaced with cardiomyocyte maintenance medium and changed every 2 days. Five to seven days post thaw, the cardiomyocytes were digested, and 5–6 µL of cell suspension was plated in fibronectin-coated MEA 48-well plates (CytoView MEA 48; Axion BioSystems, Atlanta, GA) at a density of 5–6 × 10⁴ cells per well. MEA recordings were performed 10–14 days post thaw at 37 °C. RPMI-1640 plus B27 supplement (cat. no. 17504044; Thermo Fisher Scientific, Waltham, MA) was used 2 h prior to recordings of acute drug effects.

Compound preparation

Thirteen CiPA reference drugs were chosen and purchased from Sigma-Aldrich (St. Louis, MO) or Toronto Research Chemical Inc. (North York, Canada), and five concentrations of each drug were examined ( Suppl. Table S1 ). All drugs were dissolved in DMSO at 1000× the highest test concentration, and then serially diluted in RPMI-1640 plus B27 supplement to yield the test concentrations. All compound solutions were prepared immediately before use and stored in an incubator (37 °C, 5% CO₂) until added to test wells.

Experimental protocol for spontaneously beating hiPSC-vCMs

The Maestro multiwell electrophysiology platform (Axion BioSystems) was used in this study. For each drug, five concentrations were added sequentially with 20 min of recording time for each concentration. An algorithm was used to identify the 30 most stable beats within the last 5 min of the 20-min recording, and these beats were used to measure FPD or Fridericia-corrected field potential duration (FPDc), spike amplitude (AMP), and beating period (BP) with the CiPA Analysis Tool software. The occurrence of arrhythmia-like events was marked manually through the whole 20-min recording. For clearer recognition of arrhythmia-like events, the Local Extracellular Action Potential (LEAP) assay, a functional module in the Maestro MEA system that allows acquisition of extracellular AP waveforms, was used alternatively after the 20-min FP recordings.

Experimental protocol for electrical pacing of hiPSC-vCMs

For electrical pacing, one electrode from each test well was selected, and a train of biphasic electrical stimulations (400 mV, 1 ms) was applied to the cells from low to high rates (0.5 Hz to 1.2 Hz). Three minutes of recording time were analyzed for each frequency.

Statistics

Results from patch clamp recording are presented as mean ± standard error, while other results are presented as mean ± standard deviation. Student t-test (two-tail) or one-way ANOVA with Scheffe’s test was used for statistical comparison between two or multiple groups with the software Origin 8 or GraphPad Prism 7. Statistical significance was assumed when p < 0.05.

Results

Developmental Changes in Electrophysiological Characteristics of hiPSC-vCMs

Because ion channel expression undergoes developmental changes during the differentiation of hiPSC-vCMs, it is important to investigate the time window during which their ion channels activities are stable. We first measured APs, I_Na, I_Ca, and E-4031-sensitive currents, which are most closely related to drug cardiac safety assessment,¹¹ at different time points during the course of 100 days. Typical APs of hiPSC-vCMs and statistical results of hiPSC-vCMs for days 15–100 are shown in Figure 1A . The percentage of cardiomyocytes exhibiting ventricular-like APs was greater than 90% after day 20 ( Fig. 1A ). Statistical results of APs, I_Na, I_Ca, and E-4031-sensitive currents revealed that ventricular APs and densities of ion channel currents in hiPSC-vCMs were stable after 30 days of differentiation ( Fig. 1A–1E ). At this stage, AP recordings showed that hiPSC-vCMs responded as we expected to tetrodotoxin (TTX), nifedipine, and E-4031, which are specific inhibitors of I_Na, I_Ca, and E-4031-sensitive currents, respectively. In addition, early afterdepolarization was observed after exposure to 100 nM E-4031⁷ ( Suppl. Fig. S2 ). These results suggest that after 30 days of culture, hiPSC-vCMs are suitable for drug cardiac safety assessment.

Figure 1.

Determination of suitable hiPSC-vCM age for drug assessment. (A) Typical APs recorded on hiPSC-vCMs for days 12–100, and AP statistics. (B) Left: Representative traces of I_Na; middle: I-V plots before and after 10 µM TTX; and right: current densities of I_Na for days 15–100. (C) Left: Representative traces of I_Ca; middle: I-V curves before and after 10 µM nifedipine; and right: current densities of I_Ca for days 15–100. (D) Representative traces before and after 1 µM E-4031; the difference was considered as E-4031-sensitive current. (E) Left: I-V plot; and right: tail current densities of E-403-sensitive current for days 15–100. * p < 0.05 compared with day 15; # p < 0.05 compared with day 20. AP, Action potential; APA, amplitude of action potential; APD₉₀, action potential duration at 90% repolarization; dV/dt, maximal upstroke rate; hiPSC-vCMs, human induced pluripotent stem cell–derived ventricular cardiomyocytes; I_Ca, calcium current; I_Na, sodium current; I-V, current-voltage; MDP, maximal depolarization potential; N, Number of cardiomyocytes recorded; N, number of ventricular-like APs; TTX, tetrodotoxin.

Drug Proarrhythmic Risk Analyses with Spontaneously Beating hiPSC-vCMs

To evaluate the drug proarrhythmic predictability of hiPSC-vCMs, reference validation drugs were tested using MEA with the CiPA paradigm. Thirteen CiPA validation drugs were selected, including four in the high TdP-risk category (quinidine, dofetilide, D,L-sotalol, and bepridil), four in the intermediate TdP-risk category (ondansetron, cisapride, terfenadine, and chlorpromazine), and five in the low TdP-risk category (ranolazine, mexiletine, diltiazem, nifedipine, and verapamil). The study outline is shown in Figure 2A . Representative FP signals and the four endpoints of FP (BP, FPD, FPDc, and AMP) are shown in Figure 2B . The spontaneous activities of hiPSC-vCMs are listed in Table 1 . DMSO (≤ 0.1%, v/v) had no effect on the four FP endpoints, and hiPSC-vCMs properly responded to mexiletine, nifedipine, and dofetilide, which are specific inhibitors of sodium, calcium, and hERG channels, respectively ( Suppl. Fig. S3 ). Arrhythmia-like events were detected at concentrations of dofetilide higher than 0.01 µM during FP recording, and were also evident when LEAP was induced ( Fig. 2C ). Effects of the 13 examined drugs on hiPSC-vCMs are summarized in Table 2 . Prolongations of repolarization at clinical Cmax (maximum concentration) were estimated based on the dose–response curve of each drug ( Suppl. Fig. S4 ). Using the three most pivotal predictors [drug-induced arrhythmia-like events, maximum prolongation of repolarization at concentrations tested (maximum △FPDc), and the prolongation of repolarization at clinical Cmax (△FPDc at Cmax)],¹² drugs were accurately categorized into high, intermediate, and low TdP-risk groups, with the exception of bepridil and ranolazine. Bepridil did not induce arrhythmias in hiPSC-vCMs (even at 30-fold Cmax), and both maximum △FPDc and △FPDc at Cmax were shorter than other high TdP-risk drugs. In addition, values of maximum △FPDc and △FPDc at Cmax induced by ranolazine were increased compared with other drugs in the low TdP-risk category ( Fig. 2D ).

Figure 2.

Proarrhythmic risk analysis of spontaneously beating hiPSC-vCMs cultured for 30 days. (A) Experimental outline of the study. (B) Four primary endpoints of the MEA assay. (C) Example of arrhythmia-like events during field potential recording and LEAP after exposure to 0.03 µM dofetilide. (D) Three pivotal endpoints for assessment of drug-induced proarrhythmic risk. (i) Maximum ΔFPDc observed at any drug concentration for each drug (N = 6). (ii) ΔFPDc at clinical Cmax for each drug. (iii) Drug-induced arrhythmia/quiescent. ○, Drug-induced beating cessation; ▲, drug-induced arrhythmia-like events; ×, maximum concentration detected; ΔFPDc, Fridericia-corrected field potential duration; Cmax, clinical maximum concentration; hiPSC-vCMs, human induced pluripotent stem cell–derived ventricular cardiomyocytes; LEAP, local extracellular action potential recording; MEA, multielectrode array.

Table 1.

Average Electrophysiological Parameters of Spontaneously Beating hiPSC-vCMs Obtained Using Multielectrode Array.

N	BP (ms)	FPD (ms)	FPDc (ms)	AMP (mV)
124	1706 ± 249	363 ± 81	304 ± 64	4.39 ± 3.22

AMP, Spike amplitude; BP, beat period; FPD, field potential duration; FPDc, Fridericia-corrected field potential duration; hiPSC-vCMs, human induced pluripotent stem cell–derived ventricular cardiomyocytes.

Table 2.

Summary of Effects of 13 Drugs on Spontaneously Beating hiPSC-vCMs.

TdP Risk Category	Drug	Primary Ion Current Blocked	BP	AMP	FPD	FPDc	A	Q
High	Quinidine	I_Kr, I_Na-late, I_CaL	↑	↓	↑	↑	√	√
	Dofetilide	I_Kr	↑	↓	↑	↑	√	−
	Sotalol	I_Kr	↑	↓	↑	↑	√	−
	Bepridil	I_Kr, I_CaL	−	↓	↑	↑	−	√
Intermediate	Ondansetron	I_Kr, I_CaL, I_Na-late	↑	−	↑	↑	√	−
	Cisapride	I_Kr	↑	↓	↑	↑	√	−
	Terfenadine	I_Kr, I_CaL	↓	↓	↑	↑	−	√
	Chlorpromazine	I_Kr	−	−	↑	↑	−	-
Low	Ranolazine	I_Kr, I_Na-late	−	−	↑	↑	−	√
	Mexiletine	I_Na-late	−	↓	−	−	−	√
	Diltiazem	I_CaL	↓	−	↓	↓	−	√
	Nifedipine	I_CaL	↓	−	↓	↓	−	−
	Verapamil	I_CaL, I_Kr	↓	↓	↓	↓	−	−

↑ Drug-induced significant prolongation; ↓ drug-induced significant shortening; or no effect (−) on BP, AMP, FPD, or FPDc for at least one concentration; √, drug-induced arrhythmia-like events or quiescence observed. A, Arrhythmia-like events; AMP, spike amplitude; BP, beat period; FPD, field potential duration; FPDc, Fridericia-corrected field potential duration; hiPSC-vCMs, human induced pluripotent stem cell–derived ventricular cardiomyocytes; I_Ca, calcium current; I_Na, sodium current; I_Kr, E-4031 sensitive potassium current; Q, quiescent; TdP, torsades de pointes.

Drug-Induced Prolongation of Repolarization in Electrically Paced hiPSC-vCMs

To investigate the proarrhythmic potential of drugs at a physiological heart rate (1~1.2 Hz), we explored the possibility of pacing hiPSC-vCMs with electrical stimuli and focused on drug-induced prolongation of repolarization and arrhythmia-like events.^8,12 Ten drugs, including eight high and intermediate TdP-risk drugs and two low-risk drugs, were tested with electrically paced hiPSC-vCMs at 1 and 1.2 Hz. As shown in Figure 3A , electrical pulses (400 mV for 1 ms) successfully paced hiPSC-vCMs beating at 1~1.2 Hz. When the electrical stimulus was imposed on hiPSC-vCMs, “T” waves were obviously observed ( Fig. 3B ). Moreover, “T” waves were also clearly discriminated when the FPD was prolonged after exposure to dofetilide ( Fig. 3C ). Pacing increased AMP and decreased the FPD of FPs at 1 Hz and 1.2 Hz ( Fig. 3D ). Under 1-Hz and 1.2-Hz pacing, FPD prolongations in hiPSC-vCMs were detected in response to eight high- or intermediate-risk drugs (dofetilide, sotalol, quinidine, bepridil, ondansetron, cisapride, terfenadine, and chlorpromazine), exposure at the concentration that induced obvious FPD prolongation in spontaneously beating hiPSC-vCMs (Fig. 3E and 3F and Suppl. Fig. S4 ). The FPD prolongations induced by 0.3 µM bepridil (10×Cmax) under pacing were shorter than those induced by 0.003 µM dofetilide, 30 µM sotalol, and 0.3 µM quinidine (0.1~2 × Cmax). The observed FPD prolongation induced by dofetilide occurred in a concentration-dependent manner ( Fig. 3F ). In addition, ranolazine (a low TdP-risk drug) also presented prolonged FPD at the concentration of 0.5 × Cmax under 1 Hz and 1.2 Hz pacing, compared to decreased FPD induced by diltiazem ( Suppl. Fig. S5 ). In contrast to spontaneously beating hiPSC-vCMs, pacing attenuated FPD prolongations caused by drug exposure. These results indicate that the method of electrically pacing hiPSC-vCMs with MEA is capable of detecting drug-induced repolarization prolongation at a physiological heart rate.

Figure 3.

Application of MEA assay for drug testing of electrically paced hiPSC-vCMs. (A) Experimental outline of external electrical pacing. (B) Typical field potential waveforms under 1 Hz and 1.2 Hz electrical pacing (N = 16). (C) FP signals before and after 3 nM dofetilide exposure under 1 Hz and 1.2 Hz electrical pacing. (D) Effects of electrical pacing on FPD and AMP changes. (E) Effects of electrical pacing on FPD changes induced by drugs. (F) Effects of electrical pacing on FPD changes induced by dofetilide at various concentrations. AMP, Spike amplitude; FP, field potential; FPD, field potential duration; hiPSC-vCMs, human induced pluripotent stem cell–derived ventricular cardiomyocytes; MEA, multielectrode array.

Drug-Induced Arrhythmic Events in Electrically Paced hiPSC-vCMs

To further explore the application of our MEA assay with electrically paced hiPSC-vCMs for detecting drug-induced arrhythmic events, and reveal the relationship between pacing rate and arrhythmia occurrence, 0.5–1.2 Hz pacing rates were applied to hiPSC-vCMs exposed to drug concentrations that induced arrhythmic events in spontaneously beating hiPSC-vCMs ( Fig. 4A ). For this study, we chose 0.01 µM dofetilide, 300 µM sotalol, and 0.1 µM cisapride. As shown in Figure 4B and 4C and Table 3 , at lower pacing rates (0.5 Hz and 0.7 Hz), hiPSC-vCMs beat at the electrical pacing rates, and arrhythmia-like events were observed. At 0.8–0.9 Hz, the hiPSC-vCMs presented two responses: They beat at the electrical pacing rate without any arrhythmia-like events, or beat at half of the electrical pacing rate with arrhythmia-like events. When the pacing rates increased to 1 Hz and 1.2 Hz, hiPSC-vCMs beat at half of the electrical pacing rate, and the arrhythmia-like events reappeared. No arrhythmia-like event was observed in DMSO-treated cells during pacing. These results demonstrated that the pacing rate affects the occurrence of arrhythmic-like events.

Figure 4.

Proarrhythmic risk analysis using multielectrode array of electrically paced hiPSC-vCMs. (A) Experimental outline of external electrical pacing. (B) Arrhythmic events induced by 0.01 µM dofetilide under electrical pacing from 0.5 Hz to 1.2 Hz compared with spontaneous beating (spon). (C) Arrhythmic events induced by the addition of 0.01 µM dofetilide, 100 µM sotalol, or 0.1 µM cisapride under 0.5 Hz and 1 Hz electrical pacing compared with spontaneous beating (spon). * Arrhythmia-like events; ↑ electrical stimulation position; ↓ electrical artifact; hiPSC-vCMs, human induced pluripotent stem cell–derived ventricular cardiomyocytes.

Table 3.

Effects of Drugs on Beat Rates and Arrhythmia-Like Events at Different Pacing Rates.

	No Pacing	0.5 Hz		0.7 Hz		0.9 Hz		1 Hz		1.2 Hz
	A	BR	A	BR	A	BR	A	BR	A	BR	A
Dofetilide0.01 µM	√	30	√	42	√	54	−	30	√	36	√
	√	30	√	42	√	54	−	30	√	36	√
	√	30	√	42	√	54	−	60	−	36	√
Sotalol300 µM	√	30	√	42	√	27	√	30	√	36	√
	√	30	√	42	√	54	−	30	√	36	√
	√	30	√	42	√	54	−	30	√	36	√
Cisapride0.1 µM	√	30	√	42	√	27	√	30	√	36	√
	√	30	√	42	√	54	−	30	√	36	√
	√	NC	−	42	√	54	−	30	√	36	√

√ Arrhythmia-like events observed; − no arrhythmia-like events observed; A, arrhythmia-like events; BR, beat rates; NC, not captured.

Discussion

Ventricular cardiomyocytes are a good model for drug cardiac safety assessments. Considering the different electrophysiological properties of mixed atrial and nodal cardiomyocytes,^3,13 homogeneous hiPSC-vCMs are preferred for drug TdP-risk prediction. Compared with other hiPSC-CMs, the hiPSC-vCMs used in this study were obtained through a direct ventricular differentiation protocol that yielded ventricular cardiomyocytes with more than 90% purity.⁷ Through investigation of developmental changes in APs and densities of three ion channel currents in hiPSC-vCMs after the start of differentiation, we identified a suitable time window for using these cells for drug cardiac toxicity assessment. During this time window, spontaneously beating hiPSC-vCMs correctly predicted the drug TdP risk. Even so, it should be noted that the effects induced by bepridil and ranolazine in hiPSC-vCMs were also different from those of other drugs in the same TdP-risk category, as previously reported for other hiPSC-CMs.^12,14 This discrepancy may predominantly arise from differences in ion channel gene expression levels of hiPSC-CMs compared with adult cardiomyocytes, especially higher CACNA1C and lower SCN5A gene expression in hiPSC-CMs ( Suppl. Fig. S1B ). To further promote the accuracy of drug TdP-risk prediction, especially for multichannel blocking drugs, more mature ventricular cardiomyocytes should be obtained by modifying the postdifferentiation methods (e.g., long-term pacing, changing medium components, co-culture, mechanical stress, and 3D culture).^15,16

Slow spontaneous beating of hiPSC-CMs is beneficial for their pacing at a physiological heart rate. Paced hiPSC-CMs beating at physiological rate can mimic in vivo ventricular myocytes beating under sinoatrial node innervation. When the pacing rate was close to or lower than the spontaneous beating rate, however, the internal pacemaker could not be suppressed, and doublets (a second beat within a normal beat) were induced.⁹ Compared with hiPSC-derived atrial and nodal cardiomyocytes, ventricular cardiomyocytes beat slowly.^3,4,7 The high-purity hiPSC-vCMs used here exhibit slower spontaneous beating (33 ± 5 beats per minute), which makes pacing at a physiological rate (1~1.2 Hz) feasible.

Compared with optical stimulation, electrical pacing is a simple, time-saving, and cost-effective way to pace hiPSC-CMs at a physiological heart rate because it does not require transfection of exogenous genes.¹⁷ The adverse effects mentioned earlier in hiPSC-CMs caused by electrical stimulus, such as electrophysiological remodeling and structural changes, mainly occurred with long-term, rapid, and strong-intensity electrical stimulation (i.e., 3 V for 1 week at 3 Hz, or 3 V for 6 weeks at 1.2 Hz).^18–20 One week of electrical stimulation with 3 V at 1 Hz did not, however, change the electrophysiological properties of hiPSC-CMs.¹⁸ Considering current CiPA guidance on evaluating the acute TdP risk of a drug, the electrical stimulus (400 mV) used here should only have a minor influence on the activity of hiPSC-vCMs. We applied electrical stimuli to successfully pace hiPSC-vCMs beating at 1~1.2 Hz, and reliably identified “T” waves in FP signals. FP waveforms were similar to those observed in spontaneously beating and optogenetically paced hiPSC-CMs.⁸ Our results, however, contrast with those of a previous study with no obvious “T-wave” in FP signals detected under electrical pacing;⁹ differences in MEA instrument, stimulus mode, and intensity likely explain this discrepancy in outcomes. Under electrical pacing, we reliably detected the pivotal endpoints of FP signal in drug TdP-risk assessment: prolongation of repolarization induced by high and intermediate TdP-risk drugs, and the occurrence of arrhythmic-like events.

We also noticed the rate-dependent FPD changes in this study, which were consistent with previous reports.^8,9 With the increase of the pacing rate, the drug-induced FPD prolongation was shortened. In addition, when the pacing rate increased to 0.8–0.9 Hz in this study, we started to see that the other pacing pulses failed to evoke the beating. These pulses might have fell into the effective refractory period of action potential evoked by previous pulses. At this level, if the pulse evoked beating, we didn’t observe the arrhythmia-like event. The shortened FPD at this level reduced the probability that the arrhythmia-like event generation could be the reason; it is also possible that the pulse directly interfered with the arrhythmia-like event generation. When the pacing rate was fixed at 1 Hz or 1.2 Hz, the drug-induced repolarization in hiPSC-vCMs was prolonged in a concentration-dependent manner. Similar to optogenetically paced hiPSC-CMs,⁸ our results with electrically paced hiPSC-vCMs were consistent with the outcome of drug TdP risk assessment obtained with spontaneously beating hiPSC-CMs.

With the emergence of mature hiPSC-vCMs possessing low-rate or no autonomic beating, MEA assays in electrically paced hiPSC-vCMs may still have enormous potential for drug cardiac safety assessments at a physiological rate.

Supplemental Material

Final-Reversion_Supplemental_Material_for_Assessment_of_cardiac_safety_by_Bai_et_al – Supplemental material for Assessment of Drug Proarrhythmic Potential in Electrically Paced Human Induced Pluripotent Stem Cell–Derived Ventricular Cardiomyocytes Using Multielectrode Array

Supplemental material, Final-Reversion_Supplemental_Material_for_Assessment_of_cardiac_safety_by_Bai_et_al for Assessment of Drug Proarrhythmic Potential in Electrically Paced Human Induced Pluripotent Stem Cell–Derived Ventricular Cardiomyocytes Using Multielectrode Array by Shuyun Bai, Junjie Pei, Kan Chen, Ya Zhao, Henghua Cao, Luyang Tian, Yue Ma and Haiheng Dong in SLAS Discovery

Footnotes

Supplemental material is available online with this article.

Declaration of Conflicting Interests

The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Yue Ma is the founder of Cauliscell Company.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Program on Key Basic Research Project (2019YFA0110400), Beijing Municipal Science and Technology Project (Y4DG021001), and China Postdoctoral Science Foundation.

References

Gintant

Sager

P. T.

Stockbridge

Evolution of Strategies to Improve Preclinical Cardiac Safety Testing. Nature Rev. Drug Discov. 2016, 15, 457–471.

Blinova

Stohlman

Vicente

; et al. Comprehensive Translational Assessment of Human-Induced Pluripotent Stem Cell Derived Cardiomyocytes for Evaluating Drug-Induced Arrhythmias. Toxicol. Sci. 2017, 155, 234–247.

Guo

Fiene

S. J.

; et al. High Purity Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Electrophysiological Properties of Action Potentials and Ionic Currents. Am. J. Physiol. 2011, 301, H2006–H2017.

Zhang

Wilson

G. F.

Soerens

A. G.

; et al. Functional Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells. Circ. Res. 2009, 104, e30–e41.

Yokoo

Baba

Kaichi

; et al. The Effects of Cardioactive Drugs on Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells. Biochem. Biophys. Res. Comm. 2009, 387, 482–488.

Goineau

Castagne

Electrophysiological Characteristics and Pharmacological Sensitivity of Two Lines of Human Induced Pluripotent Stem Cell Derived Cardiomyocytes Coming from Two Different Suppliers. J. Pharmacol. Toxicol. Methods. 2018, 90, 58–66.

Pei

Jiang

Bai

; et al. Chemical-Defined and Albumin-Free Generation of Human Atrial and Ventricular Myocytes from Human Pluripotent Stem Cells. Stem Cell Res. 2017, 19, 94–103.

Patel

Stohlman

Dang

; et al. Assessment of Proarrhythmic Potential of Drugs in Optogenetically Paced Induced Pluripotent Stem Cell-Derived Cardiomyocytes. Toxicol. Sci. 2019, 170, 167–179.

Zeng

Balasubramanian

Lagrutta

; et al. Response of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes to Several Pharmacological Agents When Intrinsic Syncytial Pacing Is Overcome by Acute External Stimulation. J. Pharmacol. Toxicol. Methods. 2018, 91, 18–26.

10.

Zhang

Jiang

Han

; et al. Direct Differentiation of Atrial and Ventricular Myocytes from Human Embryonic Stem Cells by Alternating Retinoid Signals. Cell Res. 2011, 21, 579–587.

11.

Kramer

Obejero-Paz

C. A.

Myatt

; et al. MICE Models: Superior to the HERG Model in Predicting Torsade de Pointes. Sci. Reports. 2013, 3, 2100.

12.

Blinova

Dang

Millard

; et al. International Multisite Study of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Drug Proarrhythmic Potential Assessment. Cell Reports. 2018, 24, 3582–3592.

13.

J. Q.

Lee

; et al. Human Embryonic Stem Cells Develop into Multiple Types of Cardiac Myocytes: Action Potential Characterization. Circ. Res. 2003, 93, 32–39.

14.

Millard

Dang

Shi

; et al. Cross-Site Reliability of Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Based Safety Assays Using Microelectrode Arrays: Results from a Blinded CiPA Pilot Study. Toxicol. Sci. 2018, 164, 550–562.

15.

Yang

Pabon

Murry

C. E.

Engineering Adolescence: Maturation of Human Pluripotent Stem Cell-Derived Cardiomyocytes. Circ. Res. 2014, 114, 511–523.

16.

Karbassi

Fenix

Marchiano

; et al. Cardiomyocyte Maturation: Advances in Knowledge and Implications for Regenerative Medicine. Nature Rev. 2020, 17, 341–359.

17.

Feldbauer

Zimmermann

Pintschovius

; et al. Channelrhodopsin-2 Is a Leaky Proton Pump. Proc. Natl. Acad. Sci. USA. 2009, 106, 12317–12322.

18.

Cui

Geng

Shi

; et al. Structural and Electrophysiological Dysfunctions Due to Increased Endoplasmic Reticulum Stress in a Long-Term Pacing Model Using Human Induced Pluripotent Stem Cell-Derived Ventricular Cardiomyocytes. Stem Cell Res. Ther. 2017, 8, 109.

19.

Geng

Wang

Cui

; et al. Rapid Electrical Stimulation Increased Cardiac Apoptosis through Disturbance of Calcium Homeostasis and Mitochondrial Dysfunction in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes. Cell. Physiol. Biochem. 2018, 47, 1167–1180.

20.

Zhang

; et al. Faradaic Reactions in Capacitive Deionization (CDI): Problems and Possibilities: A Review. Water. Res. 2018, 128, 314–330.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.72 MB